It is proposed to study the subunit structures and properties of cytochromes P-450 from mitochondria of steroid-forming organs. The subunit structures, phospholipid and heme contents of the following cytochromes P-450 will be determined: Side-chain cleavage (corpus luteum, human placenta and human adrenal), ll beta-hydroxylase and 18-hydroxylase (bovine adrenal). The amino acid compositions, nature and number of subunits and heme groups and other properties of these enzymes will be compared with each other and with the adrenal side-chain cleavage system. In the case of 18-hydroxylase, attention will be paid to the possibility that one enzyme exists for each of the two substrates (DOC and corticosterone), perhaps situated in different parts of the cortex. Attempts will be made to separate the 11 beta- and 18-hydroxylase activities which have so far been reported to copurify. Testicular microsomal P-450 will be purified and characterized to demonstrate whether two separate enzymes are present as suggested by indirect evidence. The properties of these enzymes will be determined and compared with those of the mitochondrial cytochromes P-450 (molecular weight, phospholipid and heme content). The possibility that purified side-chain cleavage P-450 can be phosphorylated by exogenous protein kinases of broad specificity as a possible prelude to more detailed studies of the role of adrenal kinase enzymes in the response to ACTH will be examined.